The long-term goals of our research are to develop inhibitors specifically targeting individual subtypes of ion channels, to determine inhibitor-channel interaction mechanisms, and to use the inhibitors to examine channel physiology and demonstrate certain therapeutic concepts. Since inward-rectifier K+ (Kir) channels play many vital physiological roles and represent increasingly important therapeutic targets, subtype-specific Kir channel inhibitors are experimentally and therapeutically valuable. The studies proposed below will be carried out with a combined approach of electrophysiology, biochemistry, and molecular biology. Previously, we discovered that a 21-residue honey bee toxin, tertiapin (TPN), inhibits renal Kir1.1 and cardiac Kir3.1/3.4 channels with nanomolar affinity. In Aim#1, we will create inhibitors specific for Kir1.1 or Kir3.1/3.4. Our preliminary studies have not only identified the channel sequence that will allow the selective targeting of various Kir subtypes but also established the prototype for such a subtype-specific inhibitor. The resulting specific inhibitors can be used in future studies to help prove the concept of new classes of medicine for treating certain cardiac diseases. Additionally, we found that piperazine - a very safe and inexpensive anthelmintic which has been shown to be anti-arrhythmic in some animal preparations - selectively inhibits strongly rectifying Kir channels such as Kir 2.1. The selectivity and safety of piperazine make it (or its derivatives) a promising candidate agent for treating certain cardiac arrhythmias. Because of this potential therapeutic value we will, in Aim#2, investigate the mechanisms by which piperazine interacts with the channel through a combination of energetic and structural studies. Furthermore, as the first step toward proving the concept, we will demonstrate that certain blocking properties of piperazine can be exploited for treating a form of short QT syndrome caused by a mutation in Kir2.1. The outcome of the proposed studies will significantly enhance our ability to decipher the physiological functions of Kir channels in a given cell type or tissue, and help in the development of effective therapeutic agents.